Nanoparticle-Conjugated Toll-Like Receptor 9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines.

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and time scale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is a critical need for improved adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improves the magnitude and duration of antibody responses when compared to soluble CpG, and results in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhances the spatiotemporal control over codelivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates humoral responses comparable to those of a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.

Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.

A TLR9 agonist synergistically enhances protective immunity induced by an Alum-adjuvanted H7N9 inactivated whole-virion vaccine.

Antigen sparing is an important strategy for pandemic vaccine development because of the limitation of worldwide vaccine production during disease outbreaks. However, several clinical studies have demonstrated that the current aluminum (Alum)-adjuvanted influenza vaccines fail to sufficiently enhance immune responses to meet licensing criteria. Here, we used pandemic H7N9 as a model virus to demonstrate that a 10-fold lower amount of vaccine antigen combined with Alum and TLR9 agonist can provide stronger protective effects than using Alum as the sole adjuvant. We found that the Alum/CpG 1018 combination adjuvant could induce more robust virus-specific humoral immune responses, including higher total IgG production, hemagglutination-inhibiting antibody activity, and neutralizing antibody titres, than the Alum-adjuvanted formulation. Moreover, this combination adjuvant shifted the immune response toward a Th1-biased immune response. Importantly, the Alum/CpG 1018-formulated vaccine could confer better protective immunity against H7N9 challenge than that adjuvanted with Alum alone. Notably, the addition of CpG 1018 to the Alum-adjuvanted H7N9 whole-virion vaccine exhibited an antigen-sparing effect without compromising vaccine efficacy. These findings have significant implications for improving Alum-adjuvanted influenza vaccines using the approved adjuvant CpG 1018 for pandemic preparedness.

TLR9 and STING agonists cooperatively boost the immune response to SARS-CoV-2 RBD vaccine through an increased germinal center B cell response and reshaped T helper responses.

Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.

Rewiring innate and adaptive immunity with TLR9 agonist to treat osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor in children and adolescent. Surgery and multidrug chemotherapy are the standard of treatment achieving 60-70% of event-free survival for localized disease at diagnosis. However, for metastatic disease, the prognosis is dismal. Exploiting immune system activation in the setting of such unfavorable mesenchymal tumors represents a new therapeutic challenge. METHODS: In immune competent OS mouse models bearing two contralateral lesions, we tested the efficacy of intralesional administration of a TLR9 agonist against the treated and not treated contralateral lesion evaluating abscopal effect. Multiparametric flow cytometry was used to evaluate changes of the tumor immune microenviroment. Experiments in immune-deficient mice allowed the investigation of the role of adaptive T cells in TLR9 agonist effects, while T cell receptor sequencing was used to assess the expansion of specific T cell clones. RESULTS: TLR9 agonist strongly impaired the growth of locally-treated tumors and its therapeutic effect also extended to the contralateral, untreated lesion. Multiparametric flow cytometry showed conspicuous changes in the immune landscape of the OS immune microenvironment upon TLR9 engagement, involving a reduction in M2-like macrophages, paralleled by increased infiltration of dendritic cells and activated CD8 T cells in both lesions. Remarkably, CD8 T cells were needed for the induction of the abscopal effect, whereas they were not strictly necessary for halting the growth of the treated lesion. T cell receptor (TCR) sequencing of tumor infiltrating CD8 T cells showed the expansion of specific TCR clones in the treated tumors and, remarkably, their selected representation in the contralateral untreated lesions, providing the first evidence of the rewiring of tumor-associated T cell clonal architectures. CONCLUSIONS: Overall these data indicate that the TLR9 agonist acts as an in situ anti-tumor vaccine, activating an innate immune response sufficient to suppress local tumor growth while inducing a systemic adaptive immunity with selective expansion of CD8 T cell clones, which are needed for the abscopal effect.

Differential immunophenotype of circulating monocytes from pregnant women in response to viral ligands.

BACKGROUND: Viral infections during pregnancy can have deleterious effects on mothers and their offspring. Monocytes participate in the maternal host defense against invading viruses; however, whether pregnancy alters monocyte responses is still under investigation. Herein, we undertook a comprehensive in vitro study of peripheral monocytes to characterize the differences in phenotype and interferon release driven by viral ligands between pregnant and non-pregnant women. METHODS: Peripheral blood was collected from third-trimester pregnant (n = 20) or non-pregnant (n = 20, controls) women. Peripheral blood mononuclear cells were isolated and exposed to R848 (TLR7/TLR8 agonist), Gardiquimod (TLR7 agonist), Poly(I:C) (HMW) VacciGrade™ (TLR3 agonist), Poly(I:C) (HMW) LyoVec™ (RIG-I/MDA-5 agonist), or ODN2216 (TLR9 agonist) for 24 h. Cells and supernatants were collected for monocyte phenotyping and immunoassays to detect specific interferons, respectively. RESULTS: The proportions of classical (CD14hiCD16-), intermediate (CD14hiCD16+), non-classical (CD14loCD16+), and CD14loCD16- monocytes were differentially affected between pregnant and non-pregnant women in response to TLR3 stimulation. The proportions of pregnancy-derived monocytes expressing adhesion molecules (Basigin and PSGL-1) or the chemokine receptors CCR5 and CCR2 were diminished in response to TLR7/TLR8 stimulation, while the proportions of CCR5- monocytes were increased. Such differences were found to be primarily driven by TLR8 signaling, rather than TLR7. Moreover, the proportions of monocytes expressing the chemokine receptor CXCR1 were increased during pregnancy in response to poly(I:C) stimulation through TLR3, but not RIG-I/MDA-5. By contrast, pregnancy-specific changes in the monocyte response to TLR9 stimulation were not observed. Notably, the soluble interferon response to viral stimulation by mononuclear cells was not diminished in pregnancy. CONCLUSIONS: Our data provide insight into the differential responsiveness of pregnancy-derived monocytes to ssRNA and dsRNA, mainly driven by TLR8 and membrane-bound TLR3, which may help to explain the increased susceptibility of pregnant women to adverse outcomes resulting from viral infection as observed during recent and historic pandemics.

Nano-particulate Toll-like Receptor 9 Agonist Potentiates the Antitumor Activity of Anti-Glypican-1 Antibody.

BACKGROUND/AIM: Monoclonal antibodies (mAbs) that target tumor antigens have recently been developed. Their antitumor activity is mainly achieved through antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as tumor-infiltrated macrophages and natural killer (NK) cells. CpG oligodeoxynucleotides (ODNs) have potent antitumor activity and are considered to increase the tumor infiltration of macrophages and NK cells; however, a completely solubilized novel CpG-schizophyllan (SPG) complex, K3-SPG, displays more potent antitumor activity. We recently reported the significant antitumor activity of anti-glypican-1 (GPC1) mAb against GPC1-positive esophageal squamous cell carcinoma (ESCC) via ADCC. The aim of this study was to evaluate the potential synergistic antitumor activity of anti-GPC1 mAb and K3-SPG and elucidate the underlying mechanisms using a xenograft model of GPC1-positive human ESCC cells. MATERIALS AND METHODS: The established human esophageal cancer cell line TE14 was subcutaneously injected into SCID mice. Xenograft mice were treated with anti-GPC1 mAb, K3-SPG, or their combination. Antitumor activity was evaluated by measuring the tumor volume. For FACS analysis, agents were administrated, and tumors were resected 1 day after the final treatment. RESULTS: Anti-GPC1 mAb or K3-SPG monotherapy showed dose-dependent antitumor activity, and combination therapy with anti-GPC1 mAb and K3-SPG showed antitumor activity (p=0.0859). Flow cytometry revealed significantly increased numbers of macrophages (p=0.0133) and of the ratio of activated NK cells/total NK cells (p=0.0058) following K3-SPG or combination therapy. CONCLUSION: Combination therapy with K3-SPG and anti-GPC1 mAb or another antitumor mAb may represent a new cancer treatment option acting via ADCC.

TLR Agonist Therapy of Metastatic Breast Cancer in Mice.

Toll-like receptor (TLR) 7/8 and 9 agonists stimulate an innate immune response that supports the development of tumor-specific immunity. Previous studies showed that either agonist individually could cure mice of small tumors and that when used in combination, they could prevent the progression of larger tumors (>300 mm 3 ). To examine whether these agents combined could control metastatic disease, syngeneic mice were challenged with the highly aggressive 66cl4 triple-negative breast tumor cell line. Treatment was not initiated until pulmonary metastases were established, as verified by bioluminescent imaging of luciferase-tagged tumor cells. Results show that combined therapy with TLR7/8 and TLR9 agonists delivered to both primary and metastatic tumor sites significantly reduced tumor burden and extended survival. The inclusion of cyclophosphamide and anti-PD-L1 resulted in optimal tumor control, characterized by a 5-fold increase in the average duration of survival.

Immunogenicity and Safety of Hepatitis B Virus (HBV) Vaccine With a Toll-Like Receptor 9 Agonist Adjuvant in HBV Vaccine-Naïve People With Human Immunodeficiency Virus.

In this international, multicenter open-label study (ACTG A5379) of HepB-CpG vaccine in people with human immunodeficiency virus (HIV) without prior hepatitis B virus (HBV) vaccination, all 68 participants achieved HBV seroprotective titers after the 3-dose series in the primary analysis. No unexpected safety issues were observed.

Engineered Toll-like Receptor Nanoagonist Binding to Extracellular Matrix Elicits Safe and Robust Antitumor Immunity.

Cancer immunotherapy, such as the Toll-like receptor (TLR) agonist including CpG oligodeoxynucleotide, has shown potency in clinical settings. However, it is still confronted with multiple challenges, which include the limited efficacy and severe adverse events caused by the rapid clearance and systemic diffusion of CpG. Here we report an improved CpG-based immunotherapy approach composed of a synthetic extracellular matrix (ECM)-anchored DNA/peptide hybrid nanoagonist (EaCpG) via (1) a tailor designed DNA template that encodes tetramer CpG and additional short DNA moieties, (2) generation of elongated multimeric CpG through rolling circle amplification (RCA), (3) self-assembly of densely packaged CpG particles composed of tandem CpG building blocks and magnesium pyrophosphate, and (4) incorporation of multiple copies of ECM binding peptide through hybridization to short DNA moieties. The structurally well-defined EaCpG shows dramatically increased intratumoral retention and marginal systemic dissemination through peritumoral administration, leading to potent antitumor immune response and subsequent tumor elimination, with minimal treatment-related toxicity. Combined with conventional standard-of-care therapies, peritumor administration of EaCpG generates systemic immune responses that lead to a curative abscopal effect on distant untreated tumors in multiple cancer models, which is superior to the unmodified CpG. Taken together, EaCpG provides a facile and generalizable strategy to simultaneously potentiate the potency and safety of CpG for combinational cancer immunotherapies.

Antibody conjugates for targeted delivery of Toll-like receptor 9 agonist to the tumor tissue.

Imiquimod, a Toll-like receptor 7 (TLR7) agonist is routinely used for topical administration in basal cell carcinoma and stage zero melanoma. Similarly, the TLR agonist Bacillus Calmette-Guérin is used for the local treatment of bladder cancer and clinical trials showed treatment efficacy of intratumoral injections with TLR9 agonists. However, when administered systemically, endosomal TLR agonists cause adverse responses due to broad immune activation. Hence, strategies for targeted delivery of TLR agonists to the tumor tissue are needed to enable the widespread use of endosomal TLR agonists in the context of tumor immunotherapy. One strategy for targeted delivery of TLR agonist is their conjugation to tumor antigen-specific therapeutic antibodies. Such antibody-TLR agonist conjugates act synergistically by inducing local TLR-mediated innate immune activation which complements the anti-tumor immune mechanisms induced by the therapeutic antibody. In this study, we explored different conjugation strategies for TLR9 agonists to immunoglobulin G (IgG). We evaluated biochemical conjugation of immunostimulatory CpG oligodesoxyribonucleotides (ODN) to the HER2-specific therapeutic antibody Trastuzumab with different cross-linkers comparing stochastic with site-specific conjugation. The physiochemical make-up and biological activities of the generated Trastuzumab-ODN conjugates were characterized in vitro and demonstrated that site-specific conjugation of CpG ODN is crucial for maintaining the antigen-binding capabilities of Trastuzumab. Furthermore, site-specific conjugate was effective in promoting anti-tumor immune responses in vivo in a pseudo-metastasis mouse model with engineered human HER2-transgenic tumor cells. In this in vivo model, co-delivery of Trastuzumab and CpG ODN in form of site-specific conjugates was superior to co-injection of unconjugated Trastuzumab, CpG ODN or stochastic conjugate in promoting T cell activation and expansion. Thereby, this study highlights that site-specific conjugation of CpG ODN to therapeutic antibodies targeting tumor markers is a feasible and more reliable approach for generation of conjugates which retain and combine the functional properties of the adjuvant and the antibody.

BAX as the mediator of C-MYC sensitizes acute lymphoblastic leukemia to TLR9 agonists.

BACKGROUND: The prognosis of B-cell acute lymphoblastic leukemia (B-ALL) has improved significantly with current first-line therapy, although the recurrence of B-ALL is still a problem. Toll-like receptor 9 (TLR9) agonists have shown good safety and efficiency as immune adjuvants. Apart from their immune regulatory effect, the direct effect of TLR9 agonists on cancer cells with TLR9 expression cannot be ignored. However, the direct effect of TLR9 agonists on B-ALL remains unknown. METHODS: We discussed the relationship between TLR9 expression and the clinical characteristics of B-ALL and explored whether CpG 685 exerts direct apoptotic effect on B-ALL without inhibiting normal B-cell function. By using western blot, co-immunoprecipitation, immunofluorescence co-localization, and chromatin immunoprecipitation, we explored the mechanism of the apoptosis-inducing effect of CpG 685 in treating B-ALL cells. By exploring the mechanism of CpG 685 on B-ALL, the predictive biomarkers of the efficacy of CpG 685 in treating B-ALL were explored. These efficiencies were also confirmed in mouse model as well as clinical samples. RESULTS: High expression of TLR9 in B-ALL patients showed good prognosis. C-MYC-induced BAX activation was the key to the effect of CpG oligodeoxynucleotides against B-ALL. C-MYC overexpression promoted P53 stabilization, enhanced Bcl-2 associated X-protein (BAX) activation, and mediated transcription of the BAX gene. Moreover, combination therapy using CpG 685 and imatinib, a BCR-ABL kinase inhibitor, could reverse resistance to CpG 685 or imatinib alone by promoting BAX activation and overcoming BCR-ABL1-independent PI3K/AKT activation. CONCLUSION: TLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. CpG 685 monotherapy might be applicable to Ph- B-ALL patients with C-MYC overexpression and without BAX deletion. CpG 685 may also serve as an effective combinational therapy against Ph+ B-ALL.

Enhanced Therapeutic Efficacy of Immunostimulatory CpG-ODN by Silencing SOCS-1 with Polysaccharide/miR-155 Complexes.

For the induction of antigen-specific immune responses, adjuvants as well as antigens are essential. CpG-ODN is a potent agonist of toll-like receptor 9 (TLR9) and is known as an adjuvant to induce cellular immune responses. We previously developed a therapeutic oligonucleotide delivery system based on the formation of a complex between schizophyllan (SPG), a kind of ß-1,3-glucan, and poly(dA), which actively delivered CpG-ODN to antigen-presenting cells (APCs) in the draining lymph nodes and induced antigen-specific immune responses. However, unfortunately, the signaling pathway of TLR9 is negatively regulated by an intracellular protein called suppressor of cytokine signaling-1 (SOCS-1), which suppresses the adjuvant effect of CpG-ODN. To solve this, we focused on microRNA-155 (miR-155), which regulates innate and autoimmune processes by targeting SOCS-1. In this study, we proposed a strategy of combining miR-155 and CpG-ODN, each complexed with SPG (denoted as SPG/miR-155 and SPG/CpG, respectively), to induce a more potent immune response. As a result, we showed that the efficient delivery of miR-155 to APCs by a complex form could induce much more potent cellular immune responses than SPG/CpG alone. Furthermore, the mice treated with the combination of SPG/miR-155 and SPG/CpG showed a long delay in tumor growth occurrence and improved survival after tumor inoculation. These results indicate the possibility of therapeutic strategies for cancer.

Immunotherapy for lung cancer combining the oligodeoxynucleotides of TLR9 agonist and TGF-ß2 inhibitor.

Tumor immunotherapies have shown promising antitumor effects, especially immune checkpoint inhibitors (ICIs). However, only 12.46% of the patients benefit from the ICIs, the rest of them shows limited effects on ICIs or even accelerates the tumor progression due to the lack of the immune cell infiltration and activation in the tumor microenvironment (TME). In this study, we administrated a combination of Toll-like receptor 9 (TLR9) agonist CpG ODN and Transforming growth factor-ß2 (TGF-ß2) antisense oligodeoxynucleotide TIO3 to mice intraperitoneally once every other day for a total of four injections, and the first injection was 24 h after LLC cell inoculation. We found that the combination induced the formation of TME toward the enrichment and activation of CD8+ T cells and NK cells, accompanied with a marked decrease of TGF-ß2. The combined therapy also effectively inhibited the tumor growth and prolonged the survival of the mice, even protected the tumor-free mice from the tumor re-challenge. Both of CpG ODN and TIO3 are indispensable, because replacing CpG ODN with TLR9 inhibitor CCT ODN showed no antitumor effect, CpG ODN or TIO3 alone did not lead to ideal antitumor results. This effect was possibly initiated by the activation of dendritic cells at the tumor site. This systemic antitumor immunotherapy with a combination of the two oligonucleotides (an immune stimulant and an immunosuppressive cytokine inhibitor) before the tumor formation may provide a novel strategy for clinical prevention of the postoperative tumor recurrence.

Universal and Translational Nanoparticulate CpG Adjuvant.

CpG, an agonist of toll-like receptor 9 (TLR9), has become a novel adjuvant that substantially potentiates cellular immunity. However, this agonist may increase systemic toxicity by diffusing into blood after administration and is difficult to be internalized by immune cells to reach TLR9 located in endosomes as a result of the characteristics of negative charge of CpG. Here, we applied a scalable and controllable flash nanocomplexation technology to prepare nanoparticulate CpG adjuvant (npCpG), CpG encapsulated in a physical cross-linking network of protamine and TPP. The nanoadjuvant could redirect CpG into draining lymph nodes to reduce systemic diffusion to improve safety. Further, a combination of npCpG and influenza H1N1 hemagglutinin antigen showed excellent humoral and cellular immunity, evoking high levels of antibodies and cytokines and inducing a great expansion of splenocytes in immunized mice. Also, the nanoadjuvant combined with ovalbumin antigen led to a potent cytotoxic T-cell response, substantially inhibited tumor growth, and improved the survival rate of mice in a melanoma model. This study showed the universal performances of npCpG in infectious disease prevention and tumor immunotherapy to demonstrate the translational potential.

Comparative genomic analysis of five freshwater cyanophages and reference-guided metagenomic data mining.

BACKGROUND: As important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications. RESULTS: Here, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu. CONCLUSIONS: We propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth. Video abstract.

CD169 (Siglec-1) as a Robust Human Cell Biomarker of Toll-Like Receptor 9 Agonist Immunotherapy.

Immunotherapy is a promising therapeutic area in cancer and chronic viral infections. An important component of immunotherapy in these contexts is the activation of innate immunity. Here we investigate the potential for CD169 (Siglec 1) expression on monocytes to serve as a robust biomarker for activation of innate immunity and, particular, as a proxy for IFN-α production. Specifically, we investigated the effects of Toll-like receptor 9 agonism with MGN1703 (lefitolimod) across experimental conditions ex vivo, in humanized mice, and in clinical trial participants. Ex vivo we observed that the percentage of classical monocytes expressing CD169 increased dramatically from 10% pre-stimulation to 97% 24 hrs after MGN1703 stimulation (p<0.0001). In humanized NOG mice, we observed prominent upregulation of the proportions of monocytes expressing CD169 after two doses of MGN1703 where 73% of classical monocytes were CD169 positive in bone marrow following MGN1703 treatment vs 19% in vehicle treated mice (p=0.0159). Finally, in a clinical trial in HIV-infected individuals receiving immunotherapy treatment with MGN1703, we observed a uniform upregulation of CD169 on monocytes after dosing with 97% of classical monocytes positive for CD169 (p=0.002). Hence, in this comprehensive evaluation ex vivo, in an animal model, and in a clinical trial, we find increases in the percentage of CD169 positive monocytes to be a reliable and robust biomarker of immune activation following TLR9 agonist treatment.

Evaluation of different combination of pam2CSK4, poly (I:C) and imiquimod enhance immune responses to H9N2 avian influenza antigen in dendritic cells and duck.

Current commercial H9 avian influenza viruses (AIVs) vaccines cannot provide satisfactory antibody titers and protective immunity against AIVs in duck. Toll like receptors (TLR) ligand as AIVs adjuvants can activate dendritic cells to improve immune responses in multiple animals, while the studies were absent in duck. Therefore, we investigated TLR ligands pam2CSK4, poly (I:C) and/or imiquimod enhance immune responses to inactivated H9N2 avian influenza antigen (H9N2 IAIV) in peripheral blood monocyte-derived dendritic cells (MoDCs) and duck. In vitro, we observed that transcription factor NF-κB, Th1/Th2 type cytokines (IFN-γ, IL-2 and IL-6) and the ability of catching H9N2 IAIV antigen were significantly up-regulated when H9N2 IAIV along with TLR ligands (pam2CSK4, poly (I:C) and imiquimod, alone or combination) in duck MoDCs. Also, the best enhancement effects were showed in combination of pam2CSK4, poly (I:C) and imiquimod group, whereas IFN-α showed no significant enhancement in all experimental groups. In vivo, the results demonstrated that the percentages of CD4+/ CD8+ T lymphocytes, the levels of Th1/Th2 type cytokines and H9N2 HI titers were significant enhanced in combination of pam2CSK4, poly (I:C) and imiquimod group. However, pam2CSK4 alone or combining with imiquimod showed no enhancement or additive effects on Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-6) and HI titers in Muscovy duck, respectively. Taken together, our results concluded that not all TLR ligands showed enhancement of immune responses to H9N2 IAIV in duck. The combination of poly (I:C), imiquimod and pam2CSK4 that can be an effectively adjuvant candidate for H9N2 AIVs inactivated vaccine in duck, which provide novel insights in explore waterfowl vaccine.

Toll-like receptor 9 agonists and combination therapies: strategies to modulate the tumour immune microenvironment for systemic anti-tumour immunity.

Over the past decade, tremendous progress has taken place in tumour immunotherapy, relying on the fast development of combination therapy strategies that target multiple immunosuppressive signaling pathways in the immune system of cancer patients to achieve a high response rate in clinical practice. Toll-like receptor 9 (TLR9) agonists have been extensively investigated as therapeutics in monotherapy or combination therapies for the treatment of cancer, infectious diseases and allergies. TLR9 agonists monotherapy shows limited efficacy in cancer patients; whereas, in combination with other therapies including antigen vaccines, radiotherapies, chemotherapies and immunotherapies exhibit great potential. Synthetic unmethylated CpG oligodeoxynucleotide (ODN), a commonly used agonist for TLR9, stimulate various antigen-presenting cells in the tumour microenvironment, which can initiate innate and adaptive immune responses. Novel combination therapy approaches, which co-deliver immunostimulatory CpG-ODN with other therapeutics, have been tested in animal models and early human clinical trials to induce anti-tumour immune responses. In this review, we describe the basic understanding of TLR9 signaling pathway; the delivery methods in most studies; discuss the key challenges of each of the above mentioned TLR9 agonist-based combination immunotherapies and provide an overview of the ongoing clinical trial results from CpG-ODN based combination therapies in cancer patients.

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays, antigen-specific antibody assays and cellular assays.

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax™), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M-052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.